Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. In principle, it would be equally revolutionary to be able to control and precisely manipulate diverse cellular processes using light. Most light-regulated proteins, however, either require engineering of precisely tethered semi-synthetic chromophores or control specific functions, such as channel opening. Thus our ability to use light to specifically perturb biological systems is impeded by the lack of a generic, genetically-encodable light-sensitive protein equivalent to GFP. We have recently demonstrated the use of a new genetically encoded light-control protein-protein interaction switch, derived from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principle be generically used to control diverse functions. We have shown that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. By translocating Rho family GTPases and their upstream activators, which control the actin cytoskeleton, we can use light to precisely reshape and direct the cell morphology of mammalian cells. We have demonstrated that this system works in yeast, Dictyostelium, and mammalian cells. Here we propose to develop the phytochrome system as a plug-and-play module that can be used for light-gated control of labeled molecules in space and time. We aim to optimize the light control instrumentation and to develop a molecular toolkit of single cell light control modules in a number of model systems. This highly flexible light-gated protein-protein interaction will provide remote control dials that will enable a new generation of perturbative, quantitative experiments in cell biology. PUBLIC HEALTH RELEVANCE: Living systems are very complex, and tools for precisely turning on and off individual molecular components would be extremely useful for dissecting how these systems function. We are developing a molecular tool in which red and infrared light can be used to control the location and activity of specific protein molecules within living cells. This tool will be a powerful resource for the entire biomedical research community, helping us to better dissect the origins of disease and to develop strategies for treating them.